RU2093525C1 - Cross-linked polyvinyl alcohol with the immobilized meta-aminophenylboronic acid as a sorbent for glycosylated hemoglobin assay by affinity method - Google Patents

Abstract

FIELD: analytical biochemistry, polymers. SUBSTANCE: invention proposes new sorbent for affinity assay of the glycosylated hemoglobin. High effectiveness of sorbent by ligand (above 20-fold use of 1 ml sorbent) is provided by the presence of cross-linked polyvinyl alcohol with the immobilized m-aminophenylboronic acid of the general formula given in description where amount of m-aminophenylboronic acid in sorbent is 0.37-1.49%, molecular mass of an active chain of cross-linked polyvinyl alcohol is 2·I0³÷3·I0³, Da, swelling coefficient is 80-100%. Sorbent can be used in medicine for diagnosis and course severity of diabetus mellitus. EFFECT: improved quality and property of sorbent. 3 tbl

Description

в качестве сорбента для аффинного определения гликозилированного гемоглобина, где количество м-АФБК в сорбенте составляет от 0,37 до 1,49% молекулярная масса активной цепи (Мс) сшитого ПВС равна 2•10³ -3•10³, коэффициент набухания (q_2m) равен 80-100.The invention relates to the chemistry of polymers, can be used in medicine for the diagnosis and assessment of the severity of diabetes mellitus, specifically to crosslinked polyvinyl alcohol (PVA) with immobilized m-aminophenylboronic acid (m-APBK) of the general formula

as the sorbent for the affinity determination of glycosylated hemoglobin, where the amount of m-ROSA in the sorbent is from 0.37 to 1.49%, the molecular weight of the active chain (MS) of the crosslinked PVA is 2 • 10 ³ -3 • 10 ³ , the swelling coefficient (q _2m ) is equal to 80-100.

 The specified compound and its properties are not described in the literature.

который используется для разделения олиго- и полирибонуклеотидов [1]
Известен сорбент аналогичного назначения агароза с иммобилизованной м-АФБК, который используется за рубежом для определения гликозилированного гемоглобина [2] Емкость агароза по лиганду составляет 5-15 мкМ м-АФБК на 1 мл сорбента.A known sorbent of a similar structure is a gel of polymethacrylic acid with immobilized m-aminophenylboronic acid (m-APBK) of the general formula

which is used to separate oligo- and polyribonucleotides [1]
A known sorbent for a similar purpose agarose with immobilized m-AFBK is used abroad to determine glycosylated hemoglobin [2]. The ligand agarose capacity is 5-15 μM m-AFBK per 1 ml of sorbent.

Химические реакции связывания полимера с м-АФБК:

Анализ разностного ИК-спектра сорбента, снятого в таблетках KBr, показал, что связывание лиганда с носителем происходит в результате взаимодействия аминогруппы м-АФБК с активированными соседством альдегидной группы двойными связями полимера, что подтверждается пояснением полос поглощения в области 708, 792, 860, 3000-3040 см^-1 (CH-ароматического кольца); 1308, 1580, 3450 см^-1 (Ar-NH-R); 3625, 3680 см^-1 (B(OH)₂). Определен элементарный состав сорбента. Вычислено, C 59,26; H 6,53; B 3,88. Найдено, C 58,76; H 6,87; B 0,12.The disadvantage of the sorbent based on agarose is that the frequency of use of one portion of the sorbent is limited to 3-6 cycles. This is an expensive sorbent [3]
The claimed technical solution for the first time proposed to use crosslinked polyvinyl alcohol (PVA) with immobilized m-aminophenylboronic acid as an sorbent for the affinity determination of glycosylated hemoglobin, where the amount of m-aminophenylboronic acid in the sorbent is from 0.37 to 1.49% molecular weight of the active chain crosslinked PVA Ms 2 • 10 ³ 3 • 10 ³ , the swelling coefficient q _2m is equal to 80 -100. General formula

Chemical reactions of polymer binding with m-APBK:

Analysis of the difference IR spectrum of the sorbent recorded in KBr tablets showed that the ligand is bound to the carrier as a result of the interaction of the m-APBK amino group with the double bonds of the polymer activated by the aldehyde group, which is confirmed by the explanation of the absorption bands in the regions 708, 792, 860, 3000 -3040 cm ^-1 (CH-aromatic ring); 1308, 1580, 3450 cm ^-1 (Ar-NH-R); 3625, 3680 cm ^-1 (B (OH) ₂ ). The elemental composition of the sorbent is determined. Calculated, C 59.26; H 6.53; B 3.88. Found, C 58.76; H 6.87; B 0.12.

Confirmation of the occurrence of a chemical reaction between an organic molecule and a polymer with the formation of a modified sorbent can serve as data from thermogravimetric analysis (TGA). According to dynamic TGA (in air), the chemical binding of m-APBA with crosslinked PVA leads to an increase in the onset temperature of decomposition of modified PVA (245 ^o C) compared to the original (210 ^o C). This can be caused by the inhibitory effect of -CH-NH-PhB (OH) ₂ fragments of the polymer network under conditions of thermal oxidative degradation.

приводит к образованию сшитого полимера, содержащего цепочку с активированными соседством с альдегидными группами двойными связями [5] В указанных условиях получено три параллельных образца сшитого полимера с характеристиками:
I q_2m 100, Mc 3,0•10³
II q_2m 90, Mc 2,6•10³
III q_2m 80, Mc 2,0•10³
Химическое связывание каждого из 3 образцов сшитого ПВС с м-АФБК, взятой в концентрациях 12-28 мг/мл, протекает по двойным связям полимера. Затем альдегидные группы полимера закрывают моноэтаноламином, а образовавшиеся азометиновые связи восстанавливают боргидридом натрия. Получают сорбент с содержанием бора по данным элементного анализа от 0,03 до 0,12% что соответствует количеству иммобилизованной м-АФБК от 0,37 до 1,49%
Выход за нижний предел (ниже 0,37%) количества м-АФБК в сорбенте ведет к уменьшению емкости сорбента по лиганду и соответственно к снижению кратности его использования. Выход за верхний предел (выше 1,49%) количеству м-АФБК в сорбенте нецелесообразен ввиду достижения максимальной (20) кратности использования сорбента притаффинном определении гликозилированного сорбента.According to the claimed crosslinked PVA with immobilized m-APBK obtained under the conditions of the known method [4] Polyvinyl alcohol is subjected to sodium periodate oxidation at 1,3-glycol groups, resulting in the formation of aldehydes that acetylate the remaining hydroxyl groups of the polymer. Subsequent additional acetalization with a trimer of glutaraldehyde (OHC- (CH ₂ ) ₃ -CH =

leads to the formation of a crosslinked polymer containing a chain with double bonds activated in the neighborhood of aldehyde groups [5] Under these conditions, three parallel samples of a crosslinked polymer with the characteristics are obtained:
I q _2m 100, Mc 3.0 • 10 ³
II q _2m 90, Mc 2.6 • 10 ³
III q _2m 80, Mc 2.0 • 10 ³
The chemical binding of each of the 3 samples of crosslinked PVA with m-APBK, taken at concentrations of 12-28 mg / ml, proceeds through double bonds of the polymer. Then, the aldehyde groups of the polymer are closed with monoethanolamine, and the resulting azomethine bonds are reduced with sodium borohydride. Get a sorbent with boron content according to elemental analysis from 0.03 to 0.12%, which corresponds to the number of immobilized m-AFBK from 0.37 to 1.49%
Going beyond the lower limit (below 0.37%) of the amount of m-APBK in the sorbent leads to a decrease in the capacity of the sorbent for the ligand and, accordingly, to a decrease in its frequency of use. Going beyond the upper limit (above 1.49%) to the amount of m-APBK in the sorbent is impractical due to the achievement of the maximum (20) frequency of use of the sorbent in the affinity determination of glycosylated sorbent.

 The invention is confirmed by examples.

 Example 1. Obtaining a sorbent.

The solution of polyvinyl alcohol in water is oxidized with sodium periodate at a concentration of 5-7 mg / ml in 1,2-glycol groups to aldehydes, followed by crosslinking due to the acetalization reaction of the residual hydroxyl groups of PVA with the formed aldehyde groups at pH 1.0, temperature 37 ^o C in within 48 hours. The resulting block is crushed and additionally acetalized with a 5% glutaraldehyde trimer solution under the same conditions. Then the sorbent is washed with distilled water until a negative reaction to aldehydes. Then 10 cm ^{3 of the} sorbent is washed with 20 ml of 0.1 M carbonate buffer, pH 9.6, and 20 ml of m-APBA solution (20 mg / ml) is added on the same buffer. Binding is carried out for 18 hours at room temperature. After that, the aldehyde groups are closed with 0.1 M monoethanolamine (pH 8.0) for 4 hours, and the resulting azomethine bonds are reduced with sodium borohydride solution (1 mg / ml, pH 7.5-8.0) for 2 hours.

The resulting sorbent is washed first with 0.1 M HCl, then with water and again with 0.001 M HCl until there is no m-APBA in the washings (due to the absence of absorption at 280 nm). The content of boron according to elemental analysis in the polymer is 0.12%, which corresponds to the content of m-APBK 1.49%
By analogy with example 1, examples 2 through 9 are performed (see table. 1).

 Example 10. Determination of glycosylated hemoglobin.

где гГб гликозилированный гемоглобин;
Л(1) и Л(2) объем фракций N 1 и N 2 в мл соответственно;
ОП₄₁₄(1) и ОП₄₁₄(2) оптическая плотность при 414 нм фракций N 1 и N 2 соответственно.To determine the glycosylated hemoglobin, the sorbent obtained under the conditions of Example 1 (1 cm ³ ) was placed in a column, washed with water and a start buffer (0.025 M sodium phosphate buffer, pH 9.0). Hemosylate is prepared by adding 3 volumes of a 0.02% sodium azide solution to 1 volume of previously washed red blood cells. Before testing, hemolysates are diluted 3 times with start buffer. Apply 1 ml of hemolysate to the sorbent. After the hemolysate enters the column, the buffer current is stopped for 15 minutes, then the column is washed with the start buffer (1 peak). Glycolized hemoglobin is eluted with an elution buffer (200 mM sorbitol, 100 mM 2-amino-2- (hydroxymethyl) -1,3-propanediol pH 8.5) peak N 2. The optical density of the fractions is determined at 414 nm. The content of glycosylated hemoglobin is determined by the formula:

where GBH is glycosylated hemoglobin;
L (1) and L (2) the volume of fractions N 1 and N 2 in ml, respectively;
OD ₄₁₄ (1) and OD ₄₁₄ (2) optical density at 414 nm of fractions N 1 and N 2, respectively.

The obtained sorbent was used to determine glycosylated hemoglobin in the blood of a healthy patient and a patient with diabetes mellitus. Each sample was tested 4 times. According to table 2, one can judge about a fairly high accuracy in determining the percentage of glycosylated hemoglobin, fluctuations in the values do not exceed 1-1.7%
With the sorbent obtained in the conditions of example 1, studies are presented to establish the frequency of use in affinity determination of glycosylated hemoglobin (see table. 3).

 The sorbent remains stable when using one portion of it in more than 20 cycles, then there is a sharp decrease in the percentage of glycosylated hemoglobin, as shown by the data in table. 3.

 Technical appraisal and economic advantages of the invention.

 The claimed sorbent based on cross-linked polyvinyl alcohol with immobilized m-aminophenylboronic acid provides more than 20-fold use of one portion of it in the affinity determination of glycosylated hemoglobin, while one portion of the known agarose sorbent is used no more than 5-6 times. The claimed sorbent is 320 times cheaper than the known one.

Claims (1)

Crosslinked polyvinyl alcohol with immobilized M-aminophenylboronic acid of the formula

where the amount of m-aminophenylboronic acid in the sorbent is 0.37
1.49% molecular weight of the active chain of crosslinked polyvinyl alcohol 2 • 10 ³ 3 • 10 ³ , the swelling coefficient is 80 100, as a sorbent for the affinity determination of glycosylated hemoglobin.

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